Methods rooted in chemical biology have contributed significantly to studies of integral membrane proteins. One recent key approach has been the application of genetic code expansion (GCE), which enables the site-specific incorporation of noncanonical amino acids (ncAAs) with defined chemical properties into proteins. Efficient GCE is challenging, especially for membrane proteins, which have specialized biogenesis and cell trafficking machinery and tend to be expressed at low levels in cell membranes. Many eukaryotic membrane proteins cannot be expressed functionally in E. coli and are most effectively studied in mammalian cell culture systems. Recent advances have facilitated broader applications of GCE for studies of membrane proteins. First, AARS/tRNA pairs have been engineered to function efficiently in mammalian cells. Second, bioorthogonal chemical reactions, including cell-friendly copper-free “click” chemistry, have enabled linkage of small-molecule probes such as fluorophores to membrane proteins in live cells. Finally, in concert with advances in GCE methodology, the variety of available ncAAs has increased dramatically, thus enabling the investigation of protein structure and dynamics by multidisciplinary biochemical and biophysical approaches. These developments are reviewed in the historical framework of the development of GCE technology with a focus on applications to studies of membrane proteins.

中文翻译：

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非经典氨基酸工具及其在膜蛋白研究中的应用


植根于化学生物学的方法为整合膜蛋白的研究做出了重大贡献。最近的一种关键方法是应用遗传密码扩展 （GCE），它能够将具有确定化学性质的非经典氨基酸 （ncAA） 位点特异性掺入蛋白质中。高效的 GCE 具有挑战性，尤其是对于膜蛋白，膜蛋白具有专门的生物发生和细胞运输机制，并且往往在细胞膜中以低水平表达。许多真核膜蛋白不能在大肠杆菌中功能性表达，并且在哺乳动物细胞培养系统中得到最有效的研究。最近的进展促进了 GCE 在膜蛋白研究中的广泛应用。首先，AARS/tRNA 对已被设计为在哺乳动物细胞中有效发挥作用。其次，生物正交化学反应，包括对细胞友好的无铜“点击”化学，使荧光团等小分子探针能够与活细胞中的膜蛋白连接。最后，随着 GCE 方法的进步，可用的 ncAA 的种类急剧增加，从而能够通过多学科生化和生物物理方法研究蛋白质结构和动力学。在 GCE 技术发展的历史框架中回顾了这些发展，重点是在膜蛋白研究中的应用。